There is a strong interest of the pharmaceutical industry to increase the effectiveness of bioactive compounds, such as protein therapeutics, by modulating or increasing their pharmacokinetic (PK) in vivo properties. This is especially true for bioactive compounds that are rapidly eliminated from the circulation by renal clearance. The kidney generally filters out molecules from circulation that have an apparent molecular weight below 60 kDa. One strategy to improve the pharmacokinetic properties of such small bioactive compounds is to simply increase their apparent molecular size (i.e. to increase their hydrodynamic radius), e.g. through the addition of non-proteinaceous polymer moieties such as polyethylene glycol polymers or sugar residues or the addition of proteinaceous polymer moieties such as globular proteins or unstructured polypeptides, such as those described in WO 2007/103515 and WO 2008/155134.
Other strategies harness the long circulation half-life of serum proteins, such as immunoglobulins and serum albumin. Serum albumin having a molecular weight of 67 kDa is the most abundant protein in plasma, present at about 50 mg/ml (0.6 mM), and has a serum half-life of 19 days in humans. Serum albumin helps to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in the plasma. There are several major small molecule binding sites in albumin that have been described.
It has been shown that non-covalent association with serum albumin can extend the half-life of short lived small molecules or polypeptides (WO 1991/001743). Polypeptides that are specifically binding to serum albumin, and that thereby can extend the in vivo half-life of other molecules coupled to them, include variants of bacterial albumin binding domains (e.g. WO 2005/097202 and WO 2009/016043), small peptides (e.g. Dennis, M. S., et al., J. Biol. Chem. 277(3), 35035-43, 2002 and WO 2001/045746) and fragments of immunoglobulins (e.g. WO 2008/043822, WO 2004/003019; WO 2008/043821; WO 2006/040153; WO 2006/122787 and WO 2004/041865). WO 2008/043822 refers to other binding proteins than fragments of immunoglobulins, such as molecules based on protein A domains, tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats and PDZ domains, which might be generated to specifically bind to serum albumin. Nevertheless, WO 2008/043822 does neither disclose the selection of designed ankyrin repeat domains with binding specificity for serum albumin (SA) nor concrete repeat sequence motifs of repeat domains that specifically bind to SA. Furthermore, it was described that the in vivo half-life of polypeptides can be prolonged by their genetic fusion to serum albumin (e.g. WO 1991/001743). Such an alteration of the in vivo half-life of drugs may positively alter their pharmacokinetic (PK) and/or pharmacodynamic (PD) properties. This is a key issue in the development of new and efficient therapeutics and disease treatment methods. There is therefore a need in the art of new ways of altering PK and/or PD of bioactive compounds.
There are, beside antibodies, novel binding proteins or binding domains that can be used to specifically bind a target molecule (e.g. Binz, H. K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005). One such novel class of binding proteins or binding domains are based on designed repeat proteins or designed repeat domains (WO 2002/020565; Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A., Nat. Biotechnol. 22, 575-582, 2004; Stumpp, M. T., Binz, H. K and Amstutz, P., Drug Discov. Today 13, 695-701, 2008). WO 2002/020565 describes how large libraries of repeat proteins can be constructed and their general application. Nevertheless, WO 2002/020565 does neither disclose the selection of repeat domains with binding specificity for SA nor concrete repeat sequence motifs of repeat domains that specifically bind to SA. Furthermore, WO 2002/020565 does not suggest that repeat domains with binding specificity for SA could be used to modulate the PK or PD of other molecules. These designed repeat domains harness the modular nature of repeat proteins and possess N-terminal and C-terminal capping modules to prevent the designed repeat domains from aggregation by shielding the hydrophobic core of the domain (Forrer, P., Stumpp, M. T., Binz, H. K. and Plückthun, A., FEBS letters 539, 2-6, 2003). These capping modules were based on the capping repeats of the natural guanine-adenine-binding protein (GA-binding protein). It was shown that the thermal and thermodynamic stability of these designed ankyrin repeat domains could be further increased by improving the C-terminal capping repeat derived from the GA-binding protein (Interlandi, G., Wetzel, S. K, Settanni, G., Plückthun, A. and Caflisch, A., J. Mol. Biol. 375, 837-854, 2008; Kramer, M. A, Wetzel, S. K., Plückthun, A., Mittl, P. R. E, and Grütter, M. G., J. Mol. Biol. 404, 381-391, 2010). The authors introduced a total of eight mutations into this capping module and extended its C-terminal helix by adding three distinct amino acids. Nevertheless, the introduction of these modifications in the C-terminal capping module resulted in a tendency of unwanted dimerization of a designed repeat domain carrying this mutated C-terminal capping module. Thus, there is a need for the generation of further optimized C-terminal capping modules or C-terminal capping repeats of ankyrin repeat domains.
Targeting SA to modulate the PK and/or PD with currently available approaches is not always effective. It has even become increasingly apparent that the modulation of the PK and/or PD of molecules by hijacking SA is complex and not yet fully understood.
Overall, a need exists for improved binding proteins with specificity for SA able to improve the PK and/PD of therapeutic relevant molecules or polypeptides for treating cancer and other pathological conditions.
The technical problem underlying the present invention is identifying novel binding proteins, such as repeat domains with binding specificity to SA, able to modify the PK and/or PD of therapeutic relevant molecules for an improved treatment of cancer and other pathological conditions. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.